Calcium-Independent Receptor a-latrotoxin, a Black Widow Spider neurotoxin which is uniquely potent stimulator of synaptic vesicle exocytosis. CIRL is a novel neuronal G protein-coupled receptor (GPCR) with unusual structure. Its large extracellular region has multiple domains homologous to cell adhesion and extracellular matrix proteins. Unlike canonical GPCR, CIRL consists of two heterologous non-covalently linked subunits that are the result of intracellular proteolytic processing of the precursor. interaction with a-latrotoxin and the results of in vitro expression experiments implicate CIRL in the regulation of secretion. The long-term goal of the proposed experiments is to study the structure of CIRL and to understand its physiological function. The specific aims of the proposed experiments are: 1. To analyze the role of GPS domain in the biosynthesis and trafficking of CIRL. The putative site of CIRL cleavage lies within a novel cysteine-rich GPCR Proteolysis Site (GPS) domain which is conserved in large orphan GPCRS. Mutation of GPS conserved residues results in the arrest of CIRL proteolysis. The hypothesis that GPS domain defines the proteolytic processing of CIRL will be tested by the analysis of deletion mutants, CIRL chimeras with another membrane protein, and soluble proteins with GPS domain. Since non-proteolyzed receptors cannot be detected at the plasma membrane, the importance of GPS domain in the intracellular trafficking of CIRL will be also addressed. 2. To study in detail the subunit structure of CIRL and the nature of the interaction between the subunits. Since CIRL is cleaved within GPS domain, the hypothesis will be tested that the complexing of subunits is based on the interaction of the two parts of this cleaved domain, the cysteine-rich box and a short peptide with a hydrophobic region. Using deletion and point mutation of CIRL, the sites critical for the subunit interaction will be deterined. 3. To analyze structural requirements for CIRL functions in secretion. Over-expression of CIRL in chromaffin cells results in the increase of their sensitivity to a-latrotoxin, and in the inhibition of calcium-evoked secretion in the permeabilized cells. To understand these phenomena, the role of different receptor domains in these effects will be analyzed, Since the cellular and molecular mechanisms of exocytosis in neurons and secretory cells differ substantially, similar expression experiments will be performed in neurons followed by the analysis of synaptic transmission. 4. To identify extracellular and intracellular CIRL-binding proteins. Several approaches will be used including affinity chromatography, yeast two-hybrid screens, and expression cloning. The search for CIRL-interacting proteins would be an important step to advance our knowledge of CIRL function and may lead to creation of drugs to treat neurological disorders.